1. Field of the Invention
The invention described herein relates to an apparatus and a method for growing suspension and adherent cells in vitro.
2. Background Art
Growing living cells in vitro is performed for a variety of purposes, including the production of cell derivatives, the preparation of viral vaccines, and the recovery of valuable cell by-products. Among the devices that have been developed for growing cells in vitro, the shell-and-tube type arrangement has become fairly common, particularly for growing suspension and adherent cells.
These devices use semipermeable tube-shaped hollow fibers (i.e., capillaries), contained within an outer shell, and configured so that fluid within a space external to the hollow fibers (i.e., an extra-capillary space) is segregated from fluid passing through the hollow fibers and their corresponding openings (i.e., lumens). Additionally, these devices usually include two manifold end chambers within the outer shell on opposite ends of the device. Each of the two lumens of a hollow fiber connects to a different end chamber. The end chambers and the extra-capillary space are separated by the semi-permeable membranes of the hollow fibers. The composition of the extra-capillary space can be controlled, to a certain extent, by the molecular weight cutoff, or pore size, of the membranes of the hollow fibers.
Typically, cells are grown in the extra-capillary space while a nutrient media is passed through the hollow fibers. The semipermeable nature of the hollow fibers allows nutrients and cell waste products to pass through the walls of the hollow fibers while blocking cells from doing the same. U.S. Pat. No. 4,391,912 to Yoshida et al. specifies a range of pore diameters to support the transfer of the nutrient medium from the intra-capillary to the extra-capillary space while blocking the entrance of cells into the intra-capillary space.
Shell-and-tube type bioreactors provide several advantages. For adherent cells, the use of several hollow fibers provides, within a relatively small volume, a large amount of surface area upon which the cells can grow. For both suspension and adherent cells, this large amount of surface area also facilitates localized distribution of nutrient media to the growing cells and ready collection of cell waste products. Shell-and-tube type bioreactors enable the growth of cells at much higher density rates than is possible with other cell culture devices. They can support cell densities greater than 108 cells per milliliter, whereas other cell culture devices are typically limited to densities around 106 cells per milliliter.
However, existing designs typically require external support systems to circulate the nutrient media through the hollow fibers. U.S. Pat. Nos. 3,883,393 to Knazek et al., 4,144,136 to Corbeil, 4,391,912 to Yoshida et al., 5,290,700 to Binot et al., and 5,955,353 to Amiot, all teach systems in which the nutrient medium is supplied to the intra-capillary spaces using pumps and associated connection tubing. These external circulating systems add considerably to the cost of using these types of shell-and-tube bioreactors.
What is needed is a shell-and-tube type apparatus to grow suspension and adherent cells that does not require an external circulating system for the nutrient media fluid.
The present invention describes an apparatus and method for growing cells. An extra-capillary space is formed between hollow fibers and an enclosed chamber. Cells are placed in the extra-capillary space to grow. A media reservoir holds cell-culture media. The cell-culture media is allowed to pass through a lumen of the hollow fibers and to pass nutrients through the walls of the hollow fibers to the cells in the extra-capillary space. Flow through the hollow fibers is produced by action of gravity when a rocking motion is imparted to the media reservoir or by action of gravity when the media reservoir is rotated about a horizontal axis.
Preferably, the hollow fibers are made of a semi-permeable material. The semi-permeable material allows: (1) nutrients and (2) metabolizing gasses to pass from the cell-culture media through the walls of the hollow fibers to the cells in the extra-capillary space, and (3) cell waste products and (4) gaseous waste products to pass from the extra-capillary space through the walls of the hollow fibers to the cell-culture media, while retaining the cells and large secreted products within the extra-capillary space.
Preferably, the media reservoir further includes a membrane permitting gas exchange between an exterior and an interior of the media reservoir.
Preferably, the media reservoir includes a first opening for accessing the interior of the media reservoir. The first opening allows: (1) fresh cell-culture media to be supplied to the media reservoir, (2) stale cell-culture media to be removed from the media reservoir, and (3) cell waste products to be removed from the media reservoir.
In one embodiment, the enclosed chamber is disposed within the media reservoir. An extra-chamber space is defined between the media reservoir and the enclosed chamber. Each hollow fiber has, at each end, a lumen open to the extra-chamber space. Preferably, the media reservoir includes a second opening for accessing the extra-capillary space. The second opening allows: (1) developing cells to be placed into the extra-capillary space, (2) mature cells to be removed from the extra-capillary space, (3) secreted products to be harvested, and (4) the cells to be treated with reagents, drugs, and/or DNA or RNA vectors.
In another embodiment, the media reservoir is configured to cause a flow of the cell-culture media between a first port and a second port. The enclosed chamber is connected between the first and second ports. Each hollow fiber has, at each end, a lumen open to the flow of the cell-culture media between the first and second ports. Preferably, the enclosed chamber includes a second opening for accessing the extra-capillary space. The second opening allows: (1) developing cells to be placed into the extra-capillary space, and (2) mature cells to be removed from the extra-capillary space.
Further features and advantages of the invention as well as the structure and operation of various embodiments of the present invention are described in detail below with reference to the accompanying drawings.